3D Colony Formation Assay for Breast Cancer

Two layer-colony forming assay was carried out as a three dimensional colony growth of human breast carcinoma cells by following the method described previously [10 (link)]. Briefly, 0.5 ml of 0.9% agar (Sea Plaque agarose, Cambrex Bio Science Rockland, Inc. Rockland, ME) in MEM α medium supplemented with 10% FBS was poured into the wells of a 12-well tissue culture plate as a bottom layer. After solidification, 25,000 MDA-231 cells transduced with 50 MOI Ad-ADRP,-FST or-LacZ were suspended in 0.5ml 0.5% agarose in MEM α medium containing 10% FBS and layered over the bottom layer. The cells were incubated at 37°C with 5% CO₂ for growth of colonies. On days 7 and 12, colony growth was evaluated by an automated phase contrast microscope equipped with Micro Analysis Suite (Olympus CKX41, Center Valley, PA). Colonies with an area greater than 5,000 μm² were counted using Micro Suite Five software. The ratio of colony growth of MDA-231 cells was determined between days 7 and 12.